The Role of Toll-Like Receptor 3 (TLR3) in Cancer ProgressionThe aim of this project is to determine the role Toll-Like Receptor 3 (TLR3) expression and TLR3 mediated responses play in the pathogenesis of multiple myeloma (MM) and colorectal cancer (CRC).Toll-like receptors (TLRs) are innate immune receptors which induce potent inflammatory responses upon detection of infection and damage. Activation of TLRs plays dual roles in tumorigenesis. TLR3 activation may promote anti-tumor effects through the induction of interferons (IFNs), which induces cell death in many cancer cells and promotes anti-cancer immunity. In contrast, activation of TLR3 may also lead to tumor-promoting inflammation through chronic production of inflammatory cytokines. Our main goal in this project is to unveil how expression and activation of TLR3 in the tumor microenvironment affects the progression of colorectal cancer (CRC) and multiple myeloma (MM). TLR3 senses double-stranded RNA (dsRNA) which is typically associated with viral infection. DsRNA analogues are applied in clinical trials in cancer immunotherapy, since they induce cancer cell death and promote anti-tumor immunity. However, TLR3 can also be activated by RNA released from dying cells in the tumor microenvironment, either during tumor growth, or as a result of chemotherapy. This may lead to tumor-promoting inflammation characterized by production of inflammatory cytokines such as TNF, IL-6 and IL-1b which are associated with poor prognosis in MM and CRC.
One main goal is to determine the effect of TLR activation in Mulitple Myeloma (MM) progression. We have not seen any effects of TLR3 activation on MM cell proliferation or survival. We have however observed that bone-marrow stromal cells (BMSC)s from MM patients express several TLRs. BMSCs often confer drug-resistance in MM cells and TLR activation in BMSCs may contribute to this resistance. BMSCs cells respond well to TLR3 activation and produce several inflammatory cytokines known to enhance the survival of MM cells. We are currently investigating which immune responses TLR3 mediates in these cells and how TLR3 activation influences MM survival and drug-resistance.
Another main goal is to determine the role of TLR3 in the progression of colorectal cancer (CRC). We have previously observed that normal and cancerous intestinal epithelial cells (IEC)s respond differently to TLR activators. Only IEC cell lines with metastatic potential are respond to dsRNA in a TLR3-dependent manner. This was surprising since TLR3 activation is known to impair proliferation and induce cell death in many cancer cells through production of IFNs. Metastatic IECs were found to be are resistent to dsRNA and failed to induce IFNs, but still induced inflammatory chemokines such as CXCL10. DsRNA was further also to promote the invasiveness of metastatic IECs in a TLR3-dependent manner, suggesting that TLR3 activation may promote metastasis. We have also discovered that TLR3 expression is atypical in these cells since it is expressed on the cell surface, rather than inside the cells in endosomes. Our results imply that metastatic IECs express surface TLR3 and can thereby sense extracellular stimuli, which triggers chemokine responses, and promotes invasiveness in these cells. Altered TLR3 expression may have implications for cancer progression and we are currently investigating the mechanisms of how TLR3 expression and signaling is regulated in metastatic IECs, and how this drives the invasiveness of IECs. We are also assessing the expression of TLRs and inflammatory genes in tissue biopsies from CRC patients at different stages to determine how expression correlates with stage and markers of prognosis.

2016

The Role of Toll-Like Receptor 3 (TLR3) in Cancer ProgressionToll-like receptors (TLR)s are important for early detection of invading pathogens, for activating innate immune responses and for initiating adaptive immunity. However, these pathways can also be harmful if dysregulated and inappropriate and prolonged activation can lead to chronic inflammatory diseases and cancer.We are investigating if TLR expression and TLR-mediated responses can affect tumor progression by exploring consequences of TLR activation on cancerous intestinal epithelial cells.
We have observed that normal and cancerous intestinal epithelial cells (IEC)s respond differently to TLR ligands. Healthy primary IEC responds poorly to TLR stimulation compared to cancerous IEC. We observed that only IEC-lines with metastatic potential (SW620, HCT116 and HT29) were responsive to the TLR3 ligand dsRNA. These cells were found to express TLR3 and upregulates TLR3 upon activation. The TLR3 ligand dsRNA potently induces the production of the chemokine CXCL10 and up-regulation of the chemokine receptor CXCR3. This is interesting because CXCR3 is upregulated in colon cancer, and expression is associated with metastasis and poor prognosis. We continued to investigate if TLR3 and TLR3-mediated responses in metastatic IECs could be linked to CXCR3 expression and the invasive properties of these cells. We compared the invasive ability of SW480 and SW620, two human IEC lines obtained from the same donor before and after tumour metastasis, respectively. Invasiveness was studied using a transwell invasion assay. Upon stimulation with the synthetic TLR3 ligand Poly(I:C), a threefold increase of invasive ability was observed in the metastatic cell line SW620, but not in its non-metastatic counterpart SW480. We further did RNA interference experiments to determine if this effect was mediated through TLR3, and found that the increased invasive ability was highly dependent on TLR3, as the effect was abolished when this receptor was knocked down. Further experiments confirmed that the increased invasiveness was a result of TLR3-mediated upregulation of CXCR3. Findings that suggest that TLR3 could have pro-tumorigenic effects in IECs through its ability to activate and up-regulate the chemokine receptor CXCR3, which further promotes migration.
We are continuing to investigate and dissect the underlining signalling pathways downstream of TLR3 that is responsible for mediating these effects. We are also aiming to confirm our results in patient samples. We are currently investigating TLR3 expression by immunohistochemistry (IHC) in biopsies from patients at different stages of colon cancer to assess the expression and localization of TLR3.